Inhibitors of oleamide hydrolase

ABSTRACT

Inhibitors of oleamide hydrolase, responsible for the hydrolysis of an endogenous sleep-inducing lipid (1, cis-9-octadecenamide) were designed and synthesized. The most potent inhibitors possess an electrophilic carbonyl group capable of reversibly forming a (thio) hemiacetal or (thio) hemiketal to mimic the transition state of a serine or cysteine protease catalyzed reaction. In particular, the tight binding α-keto ethyl ester 8 (1.4 nM) and the trifluoromethyl ketone inhibitor 12 (1.2 nM) were found to have exceptional inhibitory activity. In addition to the inhibitory activity, some of the inhibitors displayed agonist activity which resulted in the induction of sleep in laboratory animals.

SPECIFICATION

1. Field of the Invention

The invention relates to inhibitors of oleamide hydrolase and toagonists with respect to oleamide induced sleep. More particularly, theinvention relates to transition-state-mimetics and mechanism-basedoleamide derivatives which display inhibitory activity with respect tooleamide hydrolase and/or agonist activity with respect to oleamideinduced sleep.

2. Background of the Invention

Oleamide (1, cis-9-octadecenamide) is a naturally occurring brainconstituent that has been shown to accumulate and disappear underconditions of sleep deprivation and sleep recovery, respectively (Cravatet al., Science 1995, 268, 1506-1509; Lerner et al., Proc. Natl. Acad.Sci. U.S.A 1994, 91, 9505-9508; Cravatt et al., J. Am. Chem. Soc. 1996,118, 580-590). In a structurally specific manner, 1 has been shown toinduce physiological sleep in animals at nanomolar quantities wheninjected intravascularly (Cravat et al., Science 1995, 268, 1506-1509).Hydrolysis of 1 by an enzyme (oleamide hydrolase) present in the cellmembrane rapidly degrades oleamide to oleic acid (cis-9-octadecenoicacid). In an effort to isolate a regulatory agent responsible forcontrolling endogenous concentrations of 1, an integral membraneprotein, oleamide hydrolase, was found to catalyze the hydrolyticdegradation of oleamide to give oleic acid (cis-9-octadecenoic acid) andammonia (FIG. 3), neither of which demonstrate somnolescent activity(Cravat et al., Science 1995, 268, 1506-1509).

It has been found that oleamide hydrolase can be inhibited byphenylmethylsulfonyl fluoride, 4,4'-dithiodipyridine disulfide (a potentdisulfide forming reagent), and HgCl₂ (IC₅₀ =700 nM, K_(i), app =37 nM),but not by 1 mM EDTA. This suggests that a thiol is intimately involvedin the catalytic process and that the enzyme may be a cysteine amidaseor possibly a serine amidase with an active site cysteine residue.

A variety of tight binding or irreversible inhibitors of serine andcysteine proteases have been described. These include irreversibleinhibitors such as halomethyl ketones (Kettner et al., Biochemistry1978, 17, 4778-4784; Kettner et al., Thromb. Res. 1979, 14, 969-973; C.Giordano, et al., Eur. J. Med. Chem. 1992, 27, 865-873; Rauber et al.,Biochem. J. 1986, 239, 633-640; Angliker et al., Biochem. J. 1987, 241,871-875), Michael acceptors (Hanzlik et al., J. Med. Chem. 1984, 27,711-712), epoxides (C. Parkes, et al., Biochem. J. 1985, 230, 509-516),O-acyl hydroxylamines (Bromme et al., Biochem. J. 1989, 263, 861-866)and diazomethylketones (Green et al., J. Biol. Chem. 1981, 256,1923-1928) as well as reversible transition state mimetic inhibitorssuch as ketones (Mehdi, S. Bioorg. Chem. 1993, 21, 249-259), aldehydes(Westerik et al., J. Biol. Chem. 1972, 247, 8195-8197), cyclopropenones(Ando et al., J. Am. Chem. Soc. 1993, 115, 1174-1175) andelectron-deficient carbonyl compounds such as trifluoromethyl ketones(Wolfenden et al., Annu. Rev. Biophys. Bioeng. 1976, 5, 271; Gelb etal., Biochemistry 1985, 24, 1813-1817; Imperiali et al., Biochemistry1986, 25, 3760-376; Koutek et al., J. Biol. Chem. 1994, 269,22937-22940), α-keto acid derivatives (Li, Z. et al., J. Med. Chem.1993, 36, 3472-3480; Harbeson et al., J. Med. Chem. 1994, 37, 2918-2929;Peet et al., J. Med. Chem. 1990, 33, 394-407; Angelastro et al., J. Med.Chem. 1990, 33, 11-13) and tricarbonyl compounds (Wasserman et al., J.Org. Chem. 1993, 58, 4785-4787).

On the other hand, only one possibly specific inhibitor of oleamidehydrolase has been reported (IC₅₀ =3 μM at S!=0.26 K_(m)) (Maurelli etal., FEBS Lett. 1995, 377, 82-86) and only one report of aninvestigation of inhibitors of related fatty acid amidases has beendisclosed to date (Koutek et al., J. Biol. Chem. 1994, 269,22937-22940).

What is needed are highly potent inhibitors of oleamide hydrolase forinhibiting the hydrolysis of oleamide and agonists of oleamide inducedsleep.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is directed to inhibitors of oleamidehydrolase. The inhibitors are designed to interact with active sitecysteine residues within the oleamide hydrolase. The inhibitors arerapid, selective and highly potent (K_(i) =13 μM to 1 nM). Theinhibitors are useful for inhibiting the hydrolysis of oleamide, a sleepinducing factor. The inhibitors are also useful tools for furthercharacterizing the biological role of oleamide.

The inhibitors are of a type which include a head group and ahydrocarbon tail. The head group is covalently linked to the hydrocarbontail and includes electrophilic carbonyl. Preferred head groups may beselected from a group consisting of radicals represented by thefollowing structures: ##STR1##

Preferred hydrocarbon tails may be selected from a group consisting ofradicals represented by the following structures: ##STR2##

Preferred inhibitors include the following: ##STR3##

Another aspect of the invention is directed to a method for inhibitingoleamide hydrolyase with respect to the hydrolysis of oleamide. Themethod employs the act of contacting or combining the oleamide hydrolasewith an inhibitor. The inhibitor is of a type having a head group and ahydrocarbon tail covalently linked thereto. The head group includes anelectrophilic carbonyl group. Preferred head groups may be selected froma group consisting of radicals represented by the following structures:##STR4##

Preferred hydrocarbon tails may be selected from a group consisting ofradicals represented by the following structures: ##STR5##

Preferred inhibitors employed in the above method include the inhibitorsenumerated above and the following additional inhibitors: ##STR6##

Another aspect of the invention is directed to a method for inducingsleep within an oleamide sensitive animal. More particularly, thisaspect of the invention is directed to the administration to an oleamidesensitive animal of an effective dose of an agonist of oleamidehydrolase. A preferred agonist is represented by the followingstructure: ##STR7##

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates 22 inhibitors of oleamide hydrolase with respectiveinhibition constants (K_(i),app (μM); K_(m) =5±2 μM for oleamide.

FIG. 2 illustrates ¹ H NMR and ¹³ C NMR data to establish and quantitatethe addition of CD₃ OD or D₂ O to the electrophilic carbonyl in CD₃ ODor acetone-d₆. The data shows the extent of hydration and the relativeelectrophilic character of the inhibitor carbonyls. Expected trendsfollow 11>12>8>6≧4. Representative of these trends, 11 and 12 were fullyconverted to their hemiacetals in CD₃ OD, and the remaining agentsshowed diminished hemiacetal formation consistent with their expectedelectrophilic character: 11(100%), 12(100%), 8(75%), 6(48%), and 4(47%).

FIG. 3 illustrates the hydrolytic degradation of oleamide by oleamidehydrolase to give oleic acid (cis-9-octadecenoic acid) and ammonia.

FIG. 4 illustrates a dixon plot of the activity of compound 6 (Molar)against oleamide hydrolase catalyzed degradation of oleamide 1 (1/Rate(min/μM)).

FIG. 5 illustrates the effect of compound 11 (Molar) against oleamidehydrolase (Rate (μM/min/100 μL)).

FIG. 6 illustrates a Lineweaver-Burke plot of competitive oleamidehydrolase inhibition (1/ν(min/μM) by compound 12.

FIG. 7 illustrates the pH-rate dependence of oleamide hydrolase cleavageof compound 1 plot of relative rate against pH, with fit showingapparent active site pKa's of 5.4, 9.7, and 10.3. Rate maximum occurs atpH 10.0.

FIG. 8 illustrates in a) A common intermediate found in papain and othercysteine or serine proteases (O'Leary et al., Biochemistry 1974, 13,2077-2081); b) The possible mode of action for inhibitors 3-15.

FIG. 9 illustrates the chemical synthesis of intermediates andinhibitors 3, 6, 7, 8, 10, 24, and 26.

FIG. 10 illustrates the chemical synthesis of compound 27 from α-hydroxyacid 18 and shows trifluoromethyl ketone inhibitors 12, 13, 14, and 15where R=C₁₆ H₃₂ -mono-unsaturated hydrocarbon as shown in FIG. 1.

FIG. 11 illustrates the chemical synthesis of hydrated-saturatedcompound 11.

FIG. 12 illustrates the equations used to determine the pH-Ratedependence. The rate was obtained from the linear portion of the curvewhich was fit using a standard least squares procedure. These rates werereplotted against pH and fit with the shown equation by a weightednon-linear least-squares method.

DETAILED DESCRIPTION OF THE INVENTION

A series of potent transition-state-mimetic and mechanism-based oleamidehydrolase inhibitors 2-22 (FIGS. 1-2; 9, 10 and 11) are disclosed andcharacterized herein. These inhibitors are employable for exploring anddefining the roles of 1 (FIG. 1) as a prototypical member of a new classof biological signaling agents and oleamide hydrolase as a potentiallyimportant factor in its regulation.

The potency of the inhibitors was determined using an ion selectiveelectrode to measure the production of ammonia as the result of thehydrolysis of 100 μM oleamide (˜20 K_(m)) by a membrane boundpreparation of oleamide hydrolase. The K_(m) of oleamide was found to be5±2 μM. Inhibition constants were determined by the Dixon method (FIGS.4-6). Subject to solubility limitations, all inhibitors that were testedwere able to achieve 100% inhibition at high concentrations and noinhibitor exhibited polymodal inhibition behavior characteristic of twoor more separate active sites with different K_(i) s. Since thelikelihood of two or more different enzymes binding twenty-two separateinhibitors with nearly identical affinity is remote, this stronglysuggests that a single enzyme in the preparation is responsible forgreater than 90% of the observed oleamide hydrolase activity.

The most potent inhibitors (FIG. 1) possess an electrophilic carbonylgroup capable of reversibly forming a (thio) hemiacetal or (thio)hemiketal to mimic the transition state of a serine or cysteine proteasecatalyzed reaction (FIG. 8). The relative potencies of the inhibitorswere found to follow the expected electrophilic character of thereactive carbonyl cumulating in the tight binding α-keto ethyl ester 8(1.4 nM) and the trifluoromethyl ketone inhibitor 12 (1.2 nM). A similarcorrelation between carbonyl electrophilicity and binding constant hasbeen observed in inhibitors of insect juvenile hormone esterase(Linderman et al., Rev. Pestic. Toxicol. 1991, 1, 261-9) andanandaminase (Koutek et al., J. Biol. Chem. 1994, 269, 22937-22940).However, the most electrophilic member of the set, the tricarbonylinhibitor 11 bound relatively poorly at 150 nM. This behavior may be theresult of destabilizing stearic interactions between the bulkytert-butyl ester and the enzyme or may be in part due to the sp²character at C-3, which is uncharacteristic of the natural substrate.

The extent of hydration and the relative electrophilic character of theinhibitor carbonyls could be easily and accurately assessed by NMRanalysis and they were found to follow the expected trends (e.g.11>12>8>6≧4). The central carbonyl of the tricarbonyl inhibitor 11 wasfully hydrated upon preparation and characterization. The remainingagents were isolated and characterized as their carbonyl structureswithout hydration including the reactive trifluoromethylketones. ¹ H NMRand ¹³ C NMR were used to establish and quantitate the addition of CD₃OD or D₂ O to the electrophilic carbonyl in CD₃ OD and acetone-d₆,respectively (FIG. 2). Representative of these trends, 11 and 12 werefully converted to their hemiacetals in CD₃ OD, and the remaining agentsshowed diminished hemiacetal formation consistent with their expectedelectrophilic character: 11 (100%), 12 (100%), 8 (75%), 6 (48%), and 4(47%).

While the trifluoromethyl ketones 12, 13, 14 and 15 exist in aqueoussolution almost entirely as hydrates, these compounds are thought tobind to the enzyme as reversible covalent enzyme-inhibitor hemiketalcomplexes as shown in structural studies of elastase (Takahasi et al.,J. Mol. Biol. 1988, 201, 423-428) and α-chymotrypsin (Liang et al.,Biochemistry 1987, 26, 7603-7608) and kinetic studies of a series ofserine proteases (Imperiali et al., Biochemistry 1986, 25, 3760-3767)bound to peptidyl trifluoromethyl ketones. Also, though the α-ketoamides 6 and 7 are likely to exist at least in part as the sp²keto-species in solution, α-keto amides have been observed in proteaseactive sites to be completely sp³. Similarly, aldehydes in the activesite of the cysteine protease papain bind as thiohemiacetals (Mackenzieet al., Biochemistry 1986, 25, 2293-2298; Schultz et al., FEBS Lett.1975, 50, 47-49). The hypothesis that these inhibitors bind as(thio)-hemiketals rather than gem-diols (hydrated ketones) is furthersupported by the poor inhibition of oleamide hydrolase by 16, 17 and 18,despite their structural similarity to the gem-diol. We note that thoughelectrophilicity of the reactive carbonyl seems to play a large role indictating the affinity with which these inhibitors bind to oleamidehydrolase, there are likely other factors which also exert influence onthe affinity of these compounds for oleamide hydrolase. While thealdehydes 4 and α-keto amide 7 appear to be equally electrophilic, theα-keto amide binds more tightly, suggesting that there are additionalfavorable interactions being made between the enzyme and the amidefunctionality, possibly an additional hydrogen bond(s). Similarly, theα-keto ester 8 and the trifluoromethyl ketone 12 bind equally tightlydespite the the higher electrophilicity of the trifluoromethyl ketone.

Interestingly, aldehyde 5 which incorporates a carbonyl at a positionanalogous to C-2 of oleamide was found to bind five times more tightlythan 4, which incorporates the aldehyde carbonyl in the positionanalogous to the C-1 of oleamide. This was also observed with the α-ketoester series of inhibitors, where the incorporation of the electrophiliccarbonyl at C-2 versus C-1 of oleamide (8 versus 9) resulted in a 6-foldincrease in binding affinity. This distinction was not seen in theα-keto amides or trifluoromethyl ketones. In these inhibitor classes theplacement of the electrophilic carbonyl at the C-2 versus C-1 ofoleamide position provided equally effective inhibitors. This suggeststhe possibility of subtly different binding modes for carbonylpositional analogs of C-1 versus C-2 of oleamide.

These studies also reveal that oleamide hydrolase displays anapproximately ten-fold preference for fatty acid inhibitors whichcontain a cis double bond stereochemistry at the 9 position similar tothe natural substrate. This trend is seen most clearly in thetrifluoromethyl ketone series where cis double bond containing 12 isbound approximately an order of magnitude more tightly than the transdouble bond containing 14 or the saturated derivative 15.

Most of the potential irreversible inhibitors (3, 19-21) demonstrated nomeasurable time dependent inhibitory activity over the first fifteenminutes of incubation at concentrations up to their solubility limits.The chloromethyl ketone 3 gave time independent but moderate inhibition(K_(i) =0.7 μM) which is consistent with the formation of a reversible(thio) hemiketal between the putative active site cysteine and theketone (Bell et al., Advan. Phys. Org. Chem. 1966, 4, 1-29 andreferences therein) or a reversible and non-covalent enzyme-inhibitorcomplex. The presence of an adjacent chloro substituent augments theelectrophilicity of the carbonyl, favoring nucleophilic attack.2-Chlorooleic acid (20, K_(i) =0.3 μM) also appeared to bind reversibly,with its binding mode possibly similar to oleic acid (K_(i) =6 μM).Diazomethylketone 21 bound more weakly (K_(i) =18 μM).

Such observations suggest 1 may constitute a prototypical member of aclass of fatty acid primary amide biological signaling molecules inwhich the diversity and selectivity of function is derived from thelength of the alkane chain as well as the position, stereochemistry anddegree of unsaturation.

The rate of enzyme catalyzed oleamide hydrolysis was found to be pHdependent (FIG. 7) with apparent active site pK_(a) s of 5.4, 9.7 and10.3. The unusual pH-rate dependence profile, oleamide K_(m) andinhibition results for PMSF are consistent with results by Maurelli(Maurelli et al., FEBS Lett. 1995, 377, 82-86; Mackenzi et al.,Biochemistry 1986, 25, 2293-2298), suggesting that the oleamidehydrolase presented here (from rat liver membrane fractions) and theanandamide amidohydrolase (from mouse neuroblastoma cell culturemembrane fractions) may be the same enzyme, subject to inter-speciesvariation. However, in the absence of sequence data or purified enzyme,the latter of which is often difficult to achieve with integral membraneproteins, this remains to be proven. However, our results are quitedifferent from another report of anandamide amidohydrolase activitywhich exhibits rate maxima at pH 6 and 8 (Desarnaud et al., J. Biol.Chem. 1995, 270, 6030-6035) so there is also evidence to support amany-enzyme model of fatty acid amide hydrolysis in vivo. The inhibitorswere assayed at pH 10.0, the pH at which oleamide hydrolase activity isat its maximum under our assay conditions.

Agonist Activity

Another aspect of the invention is directed to a method for inducingsleep within an oleamide sensitive animal by administrating an effectivedose of an agonist of oleamide hydrolase. A preferred agonist iscompound 6. Compound 6 was dissolved in mineral oil and an effectivedose was injected into the peritoneum of a rat by intra-abdominalinjection. Sleep was monitored for the following four hours. Total sleeptime was determined by standard electrophysiological methods. Anincrease of deep slow-wave sleep (SWS) with a reduction of the wakingperiod was observed. An increase of SWS of approximately 30% and asimilar percent of reduction of waking was observed.

Inhibitor Synthesis

Many of the inhibitors were prepared from oleic acid by known proceduresor adaption of known procedures (FIG. 9). Reaction of the acid chloridederived from oleic acid (3 equivalents (COCl)₂, CH₂ Cl₂, 25° C., 3hours) with hydroxylamine or diazomethane provided 2 and 21 and directcondensation of oleic acid with hydrazine (1.1 equivalents, 2.2equivalents EDCI, 0.2 equivalent DMAP, CH₂ Cl₂, 25° C., 19 hours)provided 22. Treatment of 21 with anhydrous 1N HCl-EtOAc (25° C., 10minutes, 92%) cleanly provided 3. The aldehyde 4 (Mancuso et al., J.Org. Chem. 1978, 43, 2480-2482) along with the dimethyl acetal 16 (Marxet al., J. Med. Chem. 1989, 32, 1319-1322) could be prepared directlyfrom oleic acid as described. Trap of the enolate derived from oleicacid (LDA, THF) with CCl₄ or O₂ provided 20 (Snider et al., J. Org.Chem. 1987, 52, 307-310) and 18 (Konen et al., J. Org. Chem. 1975, 40,3253-3258) respectively, which were converted to the correspondingprimary amides 19 and 17 via acid chloride generation (3 equivalents(COCl)₂, CH₂ Cl₂, 25° C., 3 hours) and condensation with aqueous NH₄ OH.

The C-18 oleic acid based α-ketoamide 6 and α-ketoester 8 bearing anelectrophilic carbonyl at the position analogous to C-2 of oleamiderather than C-1 were prepared by oxidation of the α-hydroxyamide 17(PDC) and α-hydroxy acid 18 (Dess-Martin) followed by ethyl esterformation (FIG. 9). The corresponding α-ketoesters 9 and 10, bearing anelectrophilic carbonyl at the position analogous to C-1 of oleamide wereprepared directly from the corresponding eighteen carbon carboxylicacids, oleic and stearic acids, employing a modified Dakin-West reaction(Buchanan et al., Chem. Soc. Rev. 1988, 17, 91-109)(FIG. 9). Thea-ketoamide 7 of similar length was prepared by one carbon extension ofoleic acid available through Wolff rearrangement of 21 (cat. AgOBz,CH3OH, 25° C., 2.5 hours, 82%) to provide the methyl ester 23.Hydrolysis of the methyl ester followed by conversion of the C-19carboxylic acid 24 to the α-ketoamide followed the approach detailed for6 (FIG. 9).

The trifluoromethyl ketone inhibitors 12, 13, 14, and 15 including 13which incorporates the electrophilic carbonyl at the C-2 position of aC-18 lipid containing a 9-cis olefin were prepared in one operation byconversion of the corresponding carboxylic acids to their respectiveacid chlorides and subsequent treatment with TFAA-pyridine (Boivin etal., Tetrahedron Lett. 1992, 33, 1285-1288) (6 equivalents/8equivalents, Et₂ O, 0.75-2 hours, 54-79%), (FIG. 10). Oxidative cleavageof a-hydroxy acid 18 (Pb(OAc)₄, 1.1 equivalents, 25° C., benzene, 50 m.)yielded aldehyde 5. This was further oxidized (NaClO₂) to give acid 27which was used to prepare 13.

The tricarbonyl inhibitor 11 was prepared following the proceduresdetailed by Wasserman (Wasserman et al., Tetrahedron Lett. 1992, 33,6003-6006). Treatment of the acid chloride derived from palmitic acidwith tert-butyl (triphenylphosphoranylidene)acetate (29) in the presenceof bis(trimethylsilyl)acetamide (BSA) followed by oxone oxidationprovided 11 (FIG. 11).

Potent inhibitors of the enzyme oleamide hydrolase, responsible for thehydrolysis of an endogenous sleep-inducing lipid (1,cis-9-octadecenamide), have been developed, providing insights into themechanism of the enzyme and the fundamental basis for the development ofagents for the control and regulation of sleep.

SYNTHETIC PROTOCOLS

General

Optical rotations were measured on Perkin-Elmer 241 spectrophotometer UVand visible spectra were recorded on a Beckmann DU-70 spectrometer. ¹ Hand ¹³ C NMR spectra were recorded at 400 and 500 MHZ on Bruker AMX-400and AMX-500 spectrometer. High-resolution mass spectra (HRMS) wererecorded on a VG ZAB-ZSE mass spectrometer under fast atom bombardment(FAB) conditions. Column chromatography was carried out with silica gelof 70-230 mesh. Preparative TLC was carried out on Merck Art. 5744 (0.5mm).

Synthesis of Compound 1

Compound 1 was prepared via procedures from Cravatt et al., Science1995, 268, 1506-1509.

Synthesis of Compound 4

Compound 4 was prepared via procedures from Mancuso, A. J et al., J.Org. Chem. 1978, 43, 2480-2482

Synthesis of Compound 15

Compound 15 was prepared via procedures from Koutek, B. et al., J. Biol.Chem. 1994, 269, 22937-22940.

Synthesis of Compound 16 was prepared via procedures from Marx, M. H etal., J. Med. Chem. 1989, 32, 1319-1322.

Synthesis of Compound 18

Compound 18 was prepared via procedures from Konen et al., J. Org. Chem.1975, 40, 3253-3258.

Synthesis of Compound 20

Compound 20 was prepared via procedures from Snider et al., J. Org.Chem. 1987, 52, 307-310.

Synthesis of Compound 29

Compound 29 was prepared via procedures from Cooke et al., J. Org. Chem.1982, 47, 4955-4963.

Synthesis of N-Hydroxy-9Z-octadecenamide (2)

Oleic acid (250 μL, 0.79 mmol, 1 equivalent) was dissolved in anhydrousCH₂ Cl₂ (4 mL) and cooled to 0° C. under N₂. Oxalyl chloride (2M in CH₂Cl₂, 1.2 mL, 2.4 mmol, 3 equivalents) was added slowly. The solution waswarmed to 25° C. and allowed to stir for 3 hours in the dark. Thesolvent was removed in vacuo and the flask cooled to 0° C. Excesshydroxylamine in EtOAc (the hydrochloride salt was extracted into EtOAcfrom a 50% NaOH solution before use) was added slowly. The solvent wasremoved in vacuo and chromatography (SiO₂, 1.5×13 cm, 33-66%EtOAc-hexane gradient elution) afforded N-Hydroxy-9Z-octadecenamide 2 asa white solid (104 mg, 45%): mp 61°-62° C.; ¹ H NMR (CD₃ OD, 400 MHZ) δ5.28-5.20 (m, 2H), 2.00-1.91 (m, 6H), 1.50 (p, 2H, J=6.8 Hz), 1.22-1.19(m, 20H) , 0.80 (t, 3H, J=6.9 Hz); ¹³ C NMR (CD₃ OD, 100 MHZ) δ 173.0,130.9, 130.8, 33.8, 33.1, 30.9(2), 30.6, 30.5, 30.4, 30.33(2), 30.26,30.19, 28.2, 26.8, 23.8, 14.5; IR (film) ν_(max) 3276, 2999, 2917, 2849,1665, 1621, 1463, 1428, 1117, 1067, 968 cm⁻¹ ; FABHRMS (NBA-NaI) m/z320.2577 (C₁₈ H₃₅ NO₂ +Na⁺ requires 320.2565).

Synthesis of 1-Chloro-10Z-nonadecen-2-one (3)

A sample of 21 (347 mg, 1.13 mmol, 1 equivalent) was treated with 1M HClin EtOAc (4.0 mL, 4.0 mmol, 3.5 equivalents) for 10 minutes at 25° C.before the mixture was concentrated in vacuo. Chromatography (SiO₂, 3×13cm, 5% EtOAc-hexane) afforded 3 (328 mg, 92%) as a clear oil: ¹ H NMR(CD₃ OD, 400 MHZ) δ 5.29-5.21 (m, 2H), 4.18 (s, 2H), 2.48 (t, 2H, J=7.3Hz), 1.93 (m, 4H), 1.50 (p, 2H, J=7.1 Hz), 1.31-1.21 (m, 20H) , 0.81 (t,3H, J=6.8 Hz); ¹³ C NMR (CD₃ OD, 100 MHZ δ 204.5, 130.9, 130.8, 49.3,40.3, 33.1, 30.9, 30.8, 30.6, 30.5, 30.40, 30.37, 30.19, 30.17(2), 28.1,24.6, 23.8, 14.5; IR (film) ν_(max) 2925, 2854, 1722, 1463, 1403, 1260,1101, 796, 723 cm⁻¹ ; FABHRMS (NBA) m/z 315.2468 (C₁₉ H₃₅ OCl+H⁺requires 315.2455).

Synthesis of 8Z-Heptadecenal (5)

A solution of 18 (120 mg, 0.40 mmol, 1 equivalent) in anhydrous benzene(1.6 mL) at 25° C. under N₂ was treated with Pb(OAc)₄ (197 mg, 0.44mmol, 1.1 equivalents) and the reaction mixture was stirred for 50minutes. Water (2 mL) was added and the aqueous layer was extracted withEtOAc (6×2 mL). The organic layers were dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 2×13 cm, 1-5% EtOAc-hexanegradient elution) afforded 5 (68 mg, 67%) as a clear oil. Spectralproperties agree with those described in the literature (Doleshall etal., Tetrahedron Lett. 1977, 381-382; Kemp et al., J. Am. Oil Chem. Soc.1975, 52, 300-302).

Synthesis of 2-Oxo-9Z-octadecenamide (6).

A solution of 17 (8 mg, 0.027 mmol, 1 equivalent) in anhydrous DMF (0.13mL) under Ar was treated with PDC (51 mg, 0.13 mmol, 5 equivalents) andthe reaction mixture was stirred for 1 hour at 25° C. The crude reactionwas treated with H₂ O (2 mL) and the aqueous layer was extracted withEt₂ O (4×2 mL). The organic layers were dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 1×3 cm, 20-66% EtOAc-hexanegradient elution) afforded 6 (6 mg, 70%) as a white solid and somerecovered starting material (2 mg, 26%). For 6: mp 85°-86° C.; ¹ H NMR(CDCl₃, 400 MHZ) δ 6.79 (br, 1H), 5.47 (br, 1H), 5.37-5.28 (m, 2H), 2.89(t, 2H, J=7.4 Hz), 2.02-1.93 (m, 4H), 1.59 (p, 2H, J=7.2 Hz), 1.39-1.24(m, 20H), 0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ 198.6,161.9, 130.1, 129.6, 36.5, 31.9, 29.7, 29.5(2), 29.3(2), 28.9(2), 27.2,27.1, 23.1, 22.7, 14.1; IR (film) ν_(max) 3391, 2915, 2850, 1716, 1668,1470, 1400, 1108 cm⁻¹ ; FABHRMS (NBA-CsI) m/z 428.1547 (C₁₈ H₃₃ NO₂ +Cs⁺requires 428.1566).

Synthesis of 2-Oxo-10Z-nonadecenamide (7)

A solution of 26 (42 mg, 0.14 mmol, 1 equivalent) in anhydrous CH₂ Cl₂(2.8 mL) at 25° C. was treated with o-Ph(CO₂)I(OAc)₃ (174 mg, 0.41 mmol,3 equivalents) and the reaction mixture was stirred for 1.5 hours. Themixture was treated with 10% aqueous NaOH (30 mL) and the aqueous layerwas extracted with EtOAc (3×30 mL). The organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. Chromatography (SiO₂, 1.5×13cm, 10-20% EtOAc-hexane gradient elution) afforded 7 (24 mg, 57%) as awhite solid: mp 69°-70° C.; ¹ H NMR (CDCl₃, 400 MHZ δ 6.82 (br, 1H),5.68 (br, 1H), 5.36-5.28 (m, 2H), 2.88 (t, 2H, J=7.4 Hz), 1.98 (m, 4H),1.58 (p, 2H, J=7.0 Hz), 1.28-1.24 (m, 20H), 0.85 (t, 3H, J=6.9 Hz); ¹³ CNMR (CDCl₃, 100 MHZ δ 198.7, 162.0, 130.0, 129.7, 36.5, 31.9, 29.74,29.66, 29.5, 29.3(2), 29.2, 29.1, 29.0, 27.2, 27.1, 23.1, 22.7, 14.1; IR(film) ν_(max) 3395, 3217, 2922, 2850, 1718, 1672, 1601, 1469, 1406,1115 cm⁻¹ ; FABHRMS (NBA-NaI) m/z 332.2570 (C₁₉ H₃₅ NO₂ +Na⁺ requires332.2565).

Synthesis of Ethyl 2-Oxo-9Z-octadecenoate (8)

A solution of 18 (102 mg, 0.34 mmol, 1 equivalent) in anhydrous CH₂ Cl₂(1.1 mL) at 25° C. under N₂ was treated with o-Ph(CO₂)I(OAc)₃ (287 mg,0.68 mmol, 2 equivalents) and stirred for 1 hour. The reaction mixturewas treated with 10% aqueous NaOH (20 mL) and extracted with EtOAc (3×20mL). The organic layers were dried (Na₂ SO₄), filtered, and concentratedin vacuo. The residue was dissolved in anhydrous CH₂ Cl₂ (1.5 mL) andcooled to 0° C. under N₂. Oxalyl chloride (2M in CH₂ Cl₂, 0.5 mL, 1.0mmol, 3 equivalents) was added slowly. The reaction mixture was warmedto 25° C. and was stirred in the dark for 3 hours before the solvent wasremoved in vacuo and absolute EtOH (5 mL) was added. Chromatography(SiO₂, 2×10 cm, 1-5% EtOAc-hexane) afforded 8 (36 mg, 33%) as a clearoil: ¹ H NMR (CDCl₃, 400 MHZ) δ 5.37-5.27 (m, 2H), 4.29 (q, 2H, J=7.2Hz), 2.81 (t, 2H, J=7.3 Hz), 1.98 (m, 4H), 1.61 (p, 2H, J=7.1 Hz),1.36-1.24 (m, 21H), 0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ δ194.8, 161.3, 130.1, 129.6, 62.4, 39.3, 31.9, 29.8, 29.5(2), 29.3(2),28.92, 28.86, 27.2, 27.1, 22.9, 22.7, 14.1, 14.0; IR (film) ν_(max)2925, 2854, 1729, 1462, 1260, 1056 cm⁻¹ ; FABHRMS (NBA-CsI) m/z 457.1706(C₂₀ H₃₆ O₃ +Cs⁺ Cs⁺ requires 457.1719).

Synthesis of Ethyl 2-Oxo-10Z-nonadecenoate (9)

A solution of oleic acid (100 μL, 0.32 mmol, 1 equivalent) in anhydrousTHF (0.2 mL) at 25° C. under Ar was treated with DMAP (4 mg, 0.03 mmol,0.1 equivalent), anhydrous pyridine (77 μL, 0.95 mmol, 3 equivalents),and ethyl oxalyl chloride (71 μL, 0.64 mmol, 2 equivalents). Thereaction mixture was stirred for 24 hours before additional DMAP (46 mg,0.37 mmol, 1.1 equivalents), pyridine (80 μL, 0.95 mmol, 3 equivalents),ethyl oxalyl chloride (80 μL, 0.64 mmol, 2 equivalents), and THF (0.5mL) were added. The reaction mixture was stirred at 25° C. for anadditional 24 hours and then was warmed to 40° C. for 48 hours beforethe solvent was concentrated in vacuo. Chromatography (SiO₂, 2×13 cm,0-10% EtOAc-hexane) afforded 9 (46 mg, 43%) as a clear oil: ¹ H NMR(CDCl₃, 400 MHZ) δ 5.36-5.28 (m, 2H), 4.29 (q, 2H, J=7.1 Hz), 2.80 (t,2H, J=7.3 Hz), 1.99 (m, 4H), 1.60 (m, 2H), 1.36-1.20 (m, 23H), 0.85 (t,3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ δ 194.8, 161.2, 130.0, 129.7,62.4, 39.3, 31.9, 29.7, 29.6, 29.5, 29.3(2), 29.2, 29.0, 28.9, 27.2,27.1, 22.9, 22.7, 14.1, 14.0; IR (film) ν_(max) 2925, 2854, 1730, 1465,1260, 1059 cm⁻¹ ; FABHRMS (NBA-CsI) m/z 471.1875 (C₂₁ H₃₈ O₃ +Cs⁺requires 471.1888).

Synthesis of Ethyl 2-Oxo-nonadecanoate (10)

A solution of stearic acid (101 mg, 0.36 mmol, 1 equivalent) inanhydrous THF (0.2 mL) at 25° C. under Ar was treated with DMAP (4 mg,0.03 mmol, 0.1 equivalent), anhydrous pyridine (85 μL, 1.1 mmol, 3equivalents), and ethyl oxalyl chloride (79 μL, 0.71 mmol, 2equivalents). The reaction mixture was stirred for 24 hours before thesolvent was concentrated in vacuo. Chromatography (SiO₂, 2×13 cm, 5-10%EtOAc-hexane) afforded 10 (35 mg, 30%) as a white solid: mp 43°-44° C.;¹ H NMR (CDCl₃, 400 MHZ δ 4.29 (q, 2H, J=7.2 Hz), 2.80 (t, 2H, J=7.4Hz), 1.60 (p, 2H, J=7.2 Hz), 1.35 (t, 3H, J=7.1 Hz), 1.33-1.23 (m, 28H),0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ 194.8, 161.2, 62.4,39.3, 31.9, 29.7(7), 29.6, 29.40, 29.35, 29.28, 28.9, 23.0, 22.7, 14.1,14.0; IR (film) ν_(max) 2916, 2848, 1733, 1472, 1463, 723 cm⁻¹ ; FABHRMS(NBA-NaI) m/z 363.2885 (C₂₁ H₄₀ O₃ +Na⁺ requires 363.2875).

Synthesis of tert-Butyl 3-Oxo-2,2-dihydroxyoctadecanoate (11)

A solution of 28 (161 mg, 0.26 mmol, 1 equivalent) in THF-H₂ O (2:1; 3mL) was treated with Oxone (249 mg, 0.41 mmol, 1.6 equivalents) and thereaction mixture was stirred at 25° C. for 7 hours. Water (30 mL) wasadded and the aqueous layer was extracted with EtOAc (3×30 mL). Theorganic layers were combined, dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 2×15 cm, 10-20%EtOAc-hexane gradient elution) afforded 11 (65 mg, 64%) as a whitesolid: mp 49°-51° C; ¹ H NMR (DMSO-d₆, 400 MHZ δ 6.96 (s, 2H), 2.17 (t,2H, J=7.4 Hz), 1.49-1.38 (m, 11H), 1.22 (s, 24H), 0.84 (t, 3H, J=6.8Hz); ¹³ C NMR (DMSO-d₆, 100 MHZ δ 205.6, 174.5, 94.2, 81.5, 35.6, 33.6,31.3, 29.0(3), 28.9, 28.8, 28.72, 28.70, 28.53, 28.46, 27.4(2), 24.5,22.9, 22.1, 13.9; IR (film) ν_(max) 3440, 2914, 2849, 1728, 1471, 1371,1260, 1122, 831, 718 cm⁻¹ ; FABHRMS (NBA-NaI) m/z 409.2925 (C₂₂ H₄₂ O₅+Na⁺ requires 409.2930).

Synthesis of 1,1,1-Trifluoro-10Z-nonadecen-2-one (12)

Oleic acid (100 μL, 0.32 mmol, 1 equivalent) was dissolved in anhydrousCH₂ Cl₂ (1.5 mL) and cooled to 0° C. under N₂. Oxalyl chloride (2M inCH₂ Cl₂, 0.47 mL, 0.94 mmol, 3 equivalents) was added slowly. Thereaction mixture was warmed to 25° C. and was stirred in the dark for 3hours before the solvent was removed in vacuo. Anhydrous Et₂ O (2.2 mL),trifluoroacetic anhydride (270 μL, 1.9 mmol, 6 equivalents) and pyridine(0.2 mL, 2.5 mmol, 8 equivalents) were added at 25° C. and the solutionwas stirred for 45 minutes before being cooled to 0° C. The reaction wasquenched with the addition of H₂ O (30 mL) and the aqueous layer wasextracted with CH₂ Cl₂ (3×30 mL). The organic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo. Chromatography (SiO₂, 1.5×13cm, 1% Et₃ N in 5% EtOAc-hexane) afforded 8 (75 mg, 71%) as a clear oil:¹ H NMR (CDCl₃, 400 MHZ) δ 5.37-5.28 (m, 2H), 2.68 (t, 2H, J=7.3 Hz),1.98 (m, 4H), 1.65 (p, 2H, J=7.1 Hz), 1.29-1.25 (m, 20H), 0.86 (t, 3H,J=6.9 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ 191.6 (d, J=17 Hz), 130.0, 129.5,115.6 (q, J=145 Hz), 36.3, 31.9, 29.8, 29.6, 29.5, 29.3(2), 29.1, 29.0,28.7, 27.2, 27.1, 22.7, 22.4, 14.1; IR (film) ν_(max) 2926, 2855, 1766,1467, 1404, 1261, 1208, 1153, 1039, 802, 709 cm⁻¹ ; ESIMS m/z M⁺) 334.

Synthesis of 1,1,1-Trifluoro-9Z-octadecen-2-one (13)

A solution of 27 (101 mg, 0.38 mmol, 1 equivalent) in anhydrous CH₂ Cl₂(1.8 mL) was cooled to 0° C. under N₂ and treated dropwise with oxalylchloride (2M in CH₂ Cl₂, 0.56 mL, 1.1 mmol, 3 equivalents). The reactionmixture was warmed to 25° C. and stirred for 3 hours before the solventwas removed in vacuo. Anhydrous Et₂ O (2.5 mL), trifluoroaceticanhydride (0.32 mL, 2.3 mmol, 6 equivalents), and anhydrous pyridine(0.12 mL, 1.5 mmol, 4 equivalents) were added at 25° C. and the solutionwas stirred for 2 hours before being cooled to 0° C. The reactionmixture was treated with H₂ O (30 mL) and the aqueous layer wasextracted with EtOAc (3×30 mL). The organic layers were dried (Na₂ SO₄),filtered, and concentrated in vacuo. Chromatography (SiO₂, 2×15 cm, 1%Et₃ N in 10% EtOAc-hexane) afforded 13 (65.5 mg, 54%) as a clear oil: ¹H NMR (CDCl₃, 400 MHZ δ 5.39-5.26 (m, 2H), 2.69 (t, 2H, J=7.2 Hz), 1.99(m, 4H), 1.66 (m, 2H), 1.35-1.24 (m, 18H), 0.86 (t, 3H, J=6.9 Hz); ¹³ CNMR (CDCl₃, 100 MHZ δ 191.4, 130.5, 129.1, 115.6 (q, J=146 Hz), 36.3,31.9, 29.7, 29.5, 29.3(3), 29.2, 28.3, 27.2, 26.8, 22.7, 22.3, 14.1; IR(film) ν_(max) 2926, 2855, 1765, 1462, 1209, 1154, 1024 cm⁻¹ ; ESIMS m/z(M+Na⁺) 343.

Synthesis of 1,1,1-Trifluoro-10E-nonadecen-2-one (14)

A solution of elaidic acid (204 mg, 0.72 mmol, 1 equivalent) inanhydrous CH₂ Cl₂ (3.5 mL) was cooled to 0° C. under N₂ and treated withoxalyl chloride (2M in CH₂ Cl₂, 1.1 mL, 2.2 mmol, 3 equivalents). Thereaction mixture was warmed to 25° C. and stirred for 3 hours before thesolvent was removed in vacuo. Anhydrous Et₂ O (5 mL), trifluoroaceticanhydride (0.6 mL, 4.3 mmol, 6 equivalents), and anhydrous pyridine(0.23 mL, 2.8 mmol, 4 equivalents) were added at 25° C. and the solutionwas stirred for 1 hour before being cooled to 0° C. The mixture wastreated with H₂ O (30 mL) and the aqueous layer was extracted with EtOAc(3×30 mL). The organic layers were dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 2×13 cm, 1% Et₃ N in 5-10%EtOAc-hexane gradient elution) afforded 14 (190 mg, 79%) as a clear oil:¹ H NMR (CDCl₃, 400 MHZ) δ 5.41-5.31 (m, 2H), 2.68 (t, 2H, J=7.3 Hz),1.94 (m, 4H), 1.65 (p, 2H, J=6.9 Hz), 1.28-1.24 (m, 20H), 0.86 (t, 3H,J=6.6 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ 191.5 (q, J=35 Hz), 130.6, 130.1,115.6 (q, J=291 Hz), 36.3, 32.6, 32.5, 31.9, 29.7, 29.5(2), 29.3, 29.2,29.1, 28.8, 28.7, 22.7, 22.4, 14.0; IR (film) ν_(max) 2925, 2855, 1765,1466, 1208, 1152, 967, 709 cm⁻¹ ; FABHRMS (NBA-NaI) m/z 334.2475 (C₁₉H₃₃ OF₃ --H⁺ requires 334.2484).

Synthesis of 2-Hydroxy-9Z-octadecenamide (17)

A solution of 18 (52 mg, 0.18 mnol, 1 equivalent) in anhydrous CH₂ Cl₂(0.7 mL) cooled to 0° C. under N₂ was treated with oxalyl chloride (2Min CH₂ Cl₂, 0.22 mL, 0.44 mmol, 3 equivalents). The solution was allowedto warm to 25° C. and stirred for 3 hours in the dark. The solvent wasremoved in vacuo and the acid chloride was cooled to 0° C. The samplewas treated with excess concentrated aqueous NH₄ OH. Chromatography(SiO₂, 1.5×10 cm, 66-100% EtOAc-hexane gradient elution) afforded 17 (31mg, 60%) as a white solid: mp 103°-104° C.; ¹ H NMR (CDCl₃, 400 MHZ) δ6.37 (br, 1H), 5.64 (br, 1H), 5.36-5.28 (m, 2H), 4.12 (t, 1H, J=3.8 Hz),2.66 (br, 1H), 2.02-1.94 (m, 4H), 1.86-1.77 (m, 1H), 1.68-1.59 (m, 1H),1.43-1.24 (m, 20H), 0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ176.6, 130.0, 129.7, 71.9, 34.8, 31.9, 29.7, 29.6, 29.5, 29.3(2), 29.2,29.1, 27.2, 27.1, 24.9, 22.7, 14.1; IR (film) ν_(max) 3381, 3289, 2917,2848, 1637, 1461, 1417, 1331, 1074 cm⁻¹ ; FABHRMS (NBA) m/z 298.2760(C₁₈ H₃₅ NO₂ +H⁺ requires 298.2746) .

Synthesis of 2-Chloro-9Z-octadecenamide (19)

A solution of 20 (48 mg, 0.15 mmol, 1 equivalent) in anhydrous CH₂ Cl₂(0.7 mL) cooled to 0° C. under N₂ was treated with oxalyl chloride (2Min CH₂ Cl₂, 0.23 mL, 0.46 mmol, 3 equivalents). The solution was allowedto warm to 25° C. and was stirred for 3 hours in the dark before thesolvent was removed in vacuo. The crude acid chloride was cooled to 0°C. and treated with excess concentrated aqueous NH₄ OH. Chromatography(SiO₂, 1.5×10 cm, 20-33% EtOAc-hexane gradient elution) afforded 19 (37mg, 78%) as a yellow solid: mp 49°-50° C.; ¹ H NMR (CDCl₃, 400 MHZ) δ6.49 (br, 1H), 5.92 (br, 1H), 5.36-5.27 (m, 2H), 4.29 (m, 1H), 2.12-1.86(m, 6H), 1.53-1.16 (m, 20H), 0.85 (t, 3H, J=6.9 Hz); ¹³ C NMR (CDCl₃,100 MHZ) δ 171.9, 130.1, 129.6, 60.6, 35.5, 31.9, 29.7, 29.6, 29.5,29.3(2), 29.0, 28.7, 27.2, 27.1, 25.8, 22.7, 14.1; IR (film) ν_(max)3383, 3183, 3001, 2921, 2850, 1657, 1465, 1412, 1240, 1100 cm⁻¹ ;FABHRMS (NBA) m/z 316.2415 (C₁₈ H₃₄ NOCl+H⁺ requires 316.2407).

Synthesis of 1-Diazo-10Z-nonadecen-2-one (21)

Oleic acid (1.0 mL, 3.2 mmol, 1 equivalent) was dissolved in anhydrousCH₂ Cl₂ (15 mL) under N₂. The solution was cooled to 0° C. and oxalylchloride (2M in CH₂ Cl₂, 4.8 mL, 9.6 mmol, 3 equivalents) was added. Thereaction mixture was allowed to warm to 25° C. and was stirred for 3hours in the dark. The solvent was removed in vacuo before the acidchloride was transferred to a flask with no ground glass joints andcooled to 0° C. Excess diazomethane in Et₂ O (prepared fromN-nitrosomethylurea in 50% aqueous KOH and drying over KOH pellets) wasadded. The reaction was stirred at 0° C. for 1 hour before warming to25° C. overnight. The solution was diluted with EtOAc (60 mL) and washedwith saturated aqueous NaHCO₃ (60 mL) and saturated aqueous NaCl (60mL). The organic layer was dried (Na₂ SO₄), filtered, and concentratedin vacuo. Chromatography (SiO₂, 4.0×16 cm, 5-10% EtOAc-hexane gradientelution) afforded 21 (0.89 g, 92%) as a yellow oil: ¹ H NMR (CD₃ OD, 400MHZ) δ 5.72 (br, 1H), 5.29-5.21 (m, 2H), 2.23 (m, 2H), 1.94 (m, 4H),1.50 (p, 2H, J=6.9 Hz), 1.23-1.20 (m, 20H), 0.81 (t, 3H, J=6.9 Hz); ¹³ CNMR (CD₃ OD, 100 MHZ) δ 198.8, 130.9, 130.8, 41.6, 33.1, 30.9, 30.8(2),30.6, 30.5, 30.4(2), 30.3, 30.2, 28.1(2), 26.5, 23.8, 14.5; IR (film)ν_(max) 3083, 2924, 2854, 2102, 1644, 1463, 1372, 1144 cm⁻¹ ; FABHRMS(NBA) m/z 307.2738 (C₁₉ H₃₄ N₂ O+H⁺ requires 307.2749).

Synthesis of N-Amino-9Z-octadecenamide (22)

A solution of oleic acid (250 μL, 0.79 mmol, 1 equivalent) and hydrazinemonohydrate (42 μL, 0.87 mmol, 1.1 equivalents) in anhydrous CH₂ Cl₂ (12mL) under N₂ at 0° C. was treated with EDCI (267 mg, 0.90 mmol, 1.1equivalents) and DMAP (20 mg, 0.16 mmol, 0.21 equivalent) before thereaction mixture was allowed to stir at 25° C. for 7 hours. Anotherportion of EDCI (269 mg, 0.91 mmol, 1.1 equivalents) was added and thereaction was stirred at 25° C. for an additional 12 hours before thesolvent was removed in vacuo. Chromatography (SiO₂, 3×18 cm, 20-100%EtOAc-hexane gradient elution) afforded 22 (123 mg, 52%) as a whitesolid: mp 95°-96° C.; ¹ H NMR (CDCl₃, 400 MHZ) δ 8.94 (s, 1H), 5.36-5.27(m, 2H), 2.23 (t, 2H, J=7.6 Hz), 1.98 (m, 4H), 1.63 (p, 2H, J=7.0 Hz),1.27-1.24 (m, 20H), 0.86 (t, 3H, J=6.7 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ169.7, 130.0, 129.7, 34.1, 31.9, 29.8, 29.7, 29.5, 29.3(2), 29.23,29.19, 29.12, 27.21, 27.17, 25.4, 22.7, 14.1; IR (film) ν_(max) 3201,2917, 2848, 1595, 1410, 1184, 1090, 927, 717, 671 cm⁻¹ ; FABHRMS(NBA-NaI) m/z 297.2916 (C₁₈ H₃₆ N₂ O+H⁺ requires 297.2906).

Synthesis of Methyl 10Z-Nonadecenoate (23)

A solution of silver benzoate (21.8 mg, 0.095 mmol, 0.1 equivalent) andanhydrous Et₃ N (0.19 mL, 1.36 mmol, 1.4 equivalents) was added dropwiseto a solution of 1-diazo-10Z-nonadecen-2-one (21, 298 mg, 0.97 mmol, 1equivalent) in anhydrous CH₃ OH (1.5 mL) under N₂ and the reaction wasstirred at 25° C. for 2.5 hours. The reaction mixture was diluted withEtOAc (30 mL) and washed with 1N aqueous HCl (30 mL) and saturatedaqueous NaHCO₃ (30 mL). The organic layers were dried (Na₂ SO₄),filtered, and concentrated in vacuo. Chromatography (SiO₂, 3×15 cm, 1-5%EtOAc-hexane gradient elution) afforded 23 (246 mg, 82%) as a clear oil:¹ H NMR (CDCl₃, 400 MHZ) 6 5.35-5.27 (m, 2H), 3.63 (s, 3H), 2.27 (t, 2H,J=7.6 Hz), 1.97 (m, 4H), 1.59 (p, 2H, J=7.3 Hz), 1.26-1.24 (m, 22H),0.85 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ) δ 174.3, 129.9, 129.8,51.4, 34.1, 31.9, 29.74, 29.70, 29.5, 29.3(2), 29.2(2), 29.1(2),27.2(2), 24.9, 22.6, 14.1; IR (film) ν_(max) 2925, 2854, 1744, 1465,1436, 719 cm⁻¹ ; FABHRMS (NBA-NaI) m/z 311.2969 (C₂₀ H₃₈ O₂ +H⁺ requires311.2950).

Synthesis of 10Z-Nonadecenoic Acid (24)

A solution of 23 (620 mg, 2.0 mmol, 1 equivalent) in THF--CH₃ OH--H₂ O(3:1:1; 7 mL) at 25° C. was treated with LiOH.H₂ O (250 mg, 5.96 mmol, 3equivalents) and the reaction mixture was stirred for 3 hours. Thereaction mixture was acidified with the addition of 1N aqueous HCl (60mL) and the aqueous layer was extracted with EtOAc (2×60 mL). Theorganic layers were dried (Na₂ SO₄), filtered, and concentrated invacuo. Chromatography (SiO₂, 4×15 cm, 10-100% EtOAc-hexane gradientelution) afforded 24 (510 mg, 86%) as a pale yellow oil: ¹ H NMR (CDCl₃,400 MHZ) δ 5.37-5.28 (m, 2H), 2.32 (t, 2H, J=7.5 Hz), 1.98 (m, 4H), 1.61(p, 2H, J=7.3 Hz), 1.27-1.25 (m, 22H), 0.86 (t, 3H, J=6.9 Hz); ¹³ C NMR(CDCl₃, 100 MHZ δ 180.4, 130.0, 129.8, 34.1, 31.9, 29.8, 29.7, 29.5,29.3(2), 29.2(2), 29.0(2), 27.20, 27.17, 24.6, 22.7, 14.1; IR (film)ν_(max) 2925, 2854, 1711, 1466, 1412, 1260, 1093, 1019, 938, 801, 722cm⁻¹ ; FABHRMS (NBA-NaI) m/z 319.2605 (C₁₉ H₃₆ O₂ +Na⁺ requires319.2613). This compound can alternatively be prepared by the method ofDoleshall, G. Tetrahedron Lett. 1980, 21, 4183-4186.

Synthesis of 2-Hydroxy-10Z-nonadecenoic Acid (25)

A fresh solution of LDA was prepared at -55° C. under Ar fromdiisopropylamine (0.4 mL, 2.9 mmol, 4.5 equivalents), and n-BuLi (2.3M,1.1 mL, 2.5 mmol, 4 equivalents) in anhydrous THF (2 mL). A solution of10Z-nonadecenoic acid (24, 188 mg, 0.63 mmol, 1 equivalent) andanhydrous HMPA (0.11 mL, 0.63 mmol, 1 equivalent) in THF (0.5 mL) wasadded dropwise to the LDA solution at -55° C. The reaction mixture wasallowed to warm gradually to 25° C. and was warmed at 50° C. for 30minutes. After the reaction mixture was recooled to 25° C., O₂ wasbubbled through the solution for 20 minutes. The mixture was treatedwith 1N aqueous HCl (30 mL) and the aqueous layer was extracted withEtOAc (3×30 mL). The organic layers were dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 2×13 cm, 50-100%EtOAc-hexane gradient elution) afforded 25 (96 mg, 49%) as a whitesolid: mp 53°-54° C.; ¹ H NMR (CDCl₃, 400 MHZ) δ 5.36-5.28 (m, 2H), 4.24(dd, 1H, J=7.5 Hz, 7.6 Hz), 1.98 (m, 4H) , 1.83 (m, 1H) , 1.67 (m, 1H),1.47-1.24 (m, 22H), 0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ δ179.8, 130.0, 129.7, 70.2, 34.2, 31.9, 29.8, 29.7, 29.5, 29.33,29.31(2), 29.22, 29.19, 27.20, 27.16, 24.8, 22.7, 14.1; IR (film)ν_(max) 3512, 2917, 2849, 1704, 1467, 1293, 1274, 1251, 1212, 1143,1079, 1041, 918, 726, 648 cm⁻¹ ; FABHRMS (NBA-NaI) m/z 335.2574 (C₁₉ H₃₅O₃ +Na⁺ requires 335.2562).

Synthesis of 2-Hydroxy-10Z-nonadecenamide (26)

A solution of 25 (71 mg, 0.23 mmol, 1 equivalent) in anhydrous CH₂ Cl₂(1.5 mL) under N₂ was cooled to 0° C. and treated dropwise with oxalylchloride (2M in CH₂ Cl₂, 0.34 mL, 0.68 mmol, 3 equivalents). Thereaction mixture was allowed to warm to 25° C. and was stirred for 3hours in the dark. The solvent was removed in vacuo, the residue wascooled to 0° C., and excess concentrated aqueous NH₄ OH (2 mL) wasadded. Chromatography (SiO₂, 1.5×13 cm, 50-66% EtOAc-hexane gradientelution) afforded 26 (53 mg, 75%) as a white solid: mp 101°-102° C.; ¹ HNMR (CDCl₃, 400 MHZ) δ 6.36 (br, 1H), 5.65 (br, 1H), 5.36-5.28 (m, 2H),4.12 (dd, 1H, J=7.9 Hz, 8.0 Hz), 1.99 (m, 4H), 1.81 (m, 1H), 1.63 (m,1H), 1.43-1.24 (m, 22H), 0.86 (t, 3H, J=6.9 Hz); ¹³ C NMR (CDCl₃, 100MHZ) δ 176.6, 130.0, 129.8, 71.9, 34.8, 31.9, 29.8, 29.7, 29.5, 29.4,29.3(3), 29.2, 27.20, 27.16, 24.9, 22.7, 14.1; IR (film) ν_(max) 3383,3290, 2917, 2849, 1644, 1467, 1426, 1331, 1075 cm⁻¹ ; FABHRMS (NBA-NaI)m/z 334.2731 (C₁₉ H₃₇ NO₂ +Na⁺ requires 334.2722).

Synthesis of 8Z-Heptadecenoic acid (27)

A solution of 5 (66 mg, 0.26 mmol, 1 equivalent) and 2-methyl-2-butene(1.6 mL, 15.1 mmol, 58 equivalents) in tBuOH (6.5 mL) at 25° C. under N₂was treated dropwise with a solution of NaClO₂ (80%, 208 mg, 2.3 mmol, 9equivalents) and NaH₂ PO₄.H₂ O (250 mg, 1.8 mmol, 7 equivalents) indeionized H₂ O (2.5 mL). The reaction mixture was allowed to stir for anadditional 15 minutes before being concentrated in vacuo. The residuewas treated with water (30 mL) and the aqueous layer was extracted withEtOAc (3×30 mL). The organic layers were dried (Na₂ SO₄), filtered, andconcentrated in vacuo. Chromatography (SiO₂, 2×13 cm, 10-20%EtOAc-hexane gradient elution) afforded 27 (66 mg, 95%) as a clear oil.Spectral properties agree with those described in the S literatureMiralles et al., Lipids 1995, 30, 459-466; Couderc et al., Lipids 1995,30, 691-699.

3-Oxo-2-(triphenylphosphoranylidene)octadecanoate (28)

A solution of palmitic acid (103 mg, 0.40 mmol, 1 equivalent) inanhydrous CH₂ Cl₂ (2 mL) under N₂ was cooled to 0° C. and treated withoxalyl chloride (2M in CH₂ Cl₂, 0.6 mL, 1.2 mmol, 3 equivalents). Thesolution was allowed to stir at 25° C. for 3 hours before the solventwas removed in vacuo. A solution of tert-butyl(triphenylphosphoranylidene)acetate (Cooke et al., J. Org. Chem. 1982,47, 4955-4963) 29, 167 mg, 0.44 mmol, 1.1 equivalents) andbis(trimethylsilyl)acetamide (195 μL, 0.79 mmol, 2 equivalents), inanhydrous benzene (3 mL) at 5° C. was treated dropwise with a solutionof the crude acid chloride in benzene (3 mL). The reaction mixture wasallowed to warm to 25° C. and was stirred 1.5 hours before the solventwas removed in vacuo. Chromatography (SiO₂, 2×15 cm, 10-20% EtOAc-hexanegradient elution) afforded 28 (193 mg, 78%) as a clear oil: ¹ H NMR(CDCl₃, 400 MHZ) δ 7.67-7.61 (m, 6H), 7.49-7.37 (m, 9H), 2.82 (t, 2H,J=7.6 Hz), 1.55 (p, 2H, J=7.0 Hz), 1.23-1.21 (m, 24H), 1.04 (s, 9H),0.86 (t, 3H, J=6.8 Hz); ¹³ C NMR (CDCl₃, 100 MHZ δ 197.9 (d, J=6 Hz),167.3 (d, J=13 Hz), 132.9 (d, 6C, J=9 Hz), 131.3 (3), 128.4 (d, 6C, J=12Hz), 127.4 (d, 3C, J=96 Hz), 78.4, 71.2 (d, J=114 Hz), 40.0, 31.9,29.70(8), 29.66, 29.3, 28.1(3), 25.9, 22.7, 14.1; IR (film) ν_(max)3426, 2923, 2852, 1665, 1551, 1438, 1363, 1302, 1173, 1106, 1081, 746,690 cm⁻¹ ; FABHRMS (NBA-CsI) m/z 615.3959 (C₄₀ H₅₅ O₃ P+H⁺ requires615.3967).

Determination of Binding Constants

The potency of the these compounds against oleamide hydrolysis wasevaluated using an ion-selective ammonia electrode (ATI/Orion) todirectly measure ammonia formation as the product of the reaction. AllK_(i) s except for that of oleic acid were determined by the Dixonmethod. (X intercepts of weighted linear fits of I! versus 1/Rate plotsat a constant substrate concentration were converted to K_(i) 's usingthe formula K_(i) =-X_(int) / 1+ S!/K_(m) !.) The oleic acid K_(i) wasobtained from a non-linear weighted least-squares fit of rate versussubstrate and inhibitor concentrations. The assays were done withconstant stirring in 10 mL 50 mM CAPS buffer (Sigma) adjusted to pH10.0, the pH at which the rate of enzymatically catalyzed oleamidehydrolysis is maximal. In all cases which involved Dixon analysis, thesubstrate concentration was 100 μM. The oleic acid K_(i) was determinedover a range of substrate concentrations from 10 to 100 μM. Substrateand inhibitors were dissolved in DMSO prior to addition to the 50 mMCAPS buffer, generating a final DMSO assay concentration of 1.67%.Concentrations of up to 20% DMSO exhibited only minor effects upon therate. The enzyme concentration was adjusted to produce a rate of roughly0.2 μM/min in the absence of inhibitor and the rate of ammoniaproduction observed over a period of 7 to 10 minutes.

The enzyme was used as a crude, heterogenous, membrane-containingpreparation from rat liver. Enzyme boiled for 5 minutes demonstrated noactivity. Within solubility limits, all inhibitors achieved 100%inhibition of activity at concentrations of greater than 100 K_(i). Nodetectable activity was found in the absence of added oleamide.Likewise, only a very minimal rate of ammonia production from 100 μMoleamide was detected in the absence of enzyme at this pH. This suggeststhat the catalytic oleamide hydrolysis activity observed in this crudeenzyme preparation arises from a single protein.

The K_(m) for oleamide was determined as the average K_(m) obtained fromfour independent assays. Each independent K_(m) was obtained fromweighted linear fit of data in a Lineweaver-Burke plot. A fifthconcurring K_(m) was obtained as a result of the determination of oleicacid inhibition by non-linear methods. The rate data was fit with thestandard Michaelis-Menten kinetic equation. (The equation for the rateof Ping Pong Bi Bi kinetics collapses to the simpleMichaelis-Menten-like equation when the concentration of the secondsubstrate, in this case water, is constant). In the range 30-100 μM, thereaction rate has essentially a zero order dependence on substrateconcentration.

Because we have not yet been able to determine the amount of oleamidehydrolase present in the enzyme sample, we do not present values forV_(max) here. Our inhibition data suggests that the enzyme concentrationis lower than 2 nM, since higher enzyme concentrations would have causedsignificant depletion of inhibitor in solution, causing the apparentK_(i) to be measured as E!/2 in the limiting case of E!>>K_(i). Since 1nM was the lowest inhibition constant measured, E!<2 nM.

Error values presented with K_(i) s should be considered goodness-of-fitestimates derived from propagation of errors treatment of data. They arenot necessarily an indication of reproducibility. However, in caseswhere experiments were repeated, results were within statisticalagreement as predicted by the apparent error values given here.

pH-Rate Dependence

Crude enzyme was added to a solution of 200 μM oleamide (approximatelythe solubility limit) 1 in 20 mL buffer at the appropriate pH,containing 5% DMSO. (Concentrations of up to 20% DMSO had only minimaleffect on enzyme rates.) A 50 mM sodium citrate/Bis-tris buffer was usedfor data points in the pH 4-9 range. A 50 mM Bis-tris/CAPS was used fordata points in the 8-11 range. At pH 12, the solution was assumed to beself-buffering. At periodic time intervals, 1 mL aliquots were removedand diluted with 9 mL pH 14 buffer. Ammonia concentrations were measuredwith an ion-selective ammonia electrode (Orion) connected to a 720Ameter (Orion), calibrated against known standards. The rate was obtainedfrom the linear portion of the curve which was fit using a standardleast-squares procedure. These rates were replotted against pH and fitwith the equation in FIG. 12 (Fersht, A., Enzyme Structure andMechanism; W. H. Freeman and Co.: New York, 1985, pp 157 Connors, K. A.,Binding Constants; Wiley: New York, 1987, pp 385-395) by a weightednon-linear least-squares method (Connors, K. A., Binding Constants;Wiley: New York, 1987, pp 385-395).

In cases where two pKa's are close together (less than one unit) therewill be substantial mixing of various species of enzyme present insolution. Under such conditions, manifestation of the theoretical ratemaximum for that species may never actually be observed because the mostactive species of enzyme may never reach a high degree of abundance. Itis for this reason that simple graphical methods of determination ofpKa's may disagree with the values of 9.7 and 10.3 pH units presentedhere.

Liver Plasma Membrane Prep, Large Scale (12-14 Rat Livers)

Twelve to fourteen rat livers were sectioned and placed in 300 mL of 1mM NaHCO₃. The solution of diced liver was strained and washed withadditional 300 mL of 1 mM NaHCO₃. Any conspicuous connective tissue wasremoved. The liver was transferred to a fresh 800 mL of 1 mM NaHCO₃,stirred and then transferred in 400 mL aliquots to a blender. Blendedliver aliquots were combined and filtered through 8 layers ofcheesecloth. This was diluted to 1.0 L with 1 mM NaHCO₃ and centrifugedat 6000 rpm for 20 minutes at 4° C. (Beckman JA-17 rotor). Thesupernatant was decanted, the pellets resuspended in 1 mM NaHCO₃,combined and dounce homogenized. Centrifugation, decantation andresuspension/homogenization were repeated to give a final volume ofapproximately 90 mL. The homogenate was added to 2 volume equivalents of67% sucrose, mixed thoroughly, and transferred to ultracentrifugecompatible tubes. The tubes were topped with 30% sucrose and spun at27,000 for 2 hours (SW-28 rotor). The middle yellow band was removedfrom the sucrose gradient, combined, resuspended in 1 mM NaHCO₃, anddounce homogenized. The sample was further centrifuged at 17,000 rpm for45 minutes at 4° C. (JA-17 rotor). The supernatant was removed, thepellets resuspended in 100 mM Na₂ CO₃, dounce homogenized and left onice for 30 minutes. The solution was centrifuged at 27,000 rpm for 1hour (SW-28 rotor), the supernatant was decanted and the pelletresuspended in 15 mL of 50 mM Tris HCl, pH 7.4 with 1 mM EDTA, andhomogenized with a dounce homogenizer. This material was divided intomultiple aliquots and frozen at -78° C. until use. Each enzyme samplewas frozen once only.

What is claimed is:
 1. An inhibitor of oleamide hydrolase, saidinhibitor comprising a head group and a hydrocarbon tail covalentlylinked to said head group, wherein said head group includes anelectrophilic carbonyl and is selected from a group consisting ofradicals represented by the following structures: ##STR8## and whereinsaid hydrocarbon tail is selected from a group consisting of radicalsrepresented by the following structures: ##STR9##
 2. An inhibitor ofoleamide hydrolase as described in claim 1 represented by the followingstructure: ##STR10##
 3. An inhibitor of oleamide hydrolase as describedin claim 1 represented by the following structure: ##STR11##
 4. Aninhibitor of oleamide hydrolase as described in claim 1 represented bythe following structure: ##STR12##
 5. An inhibitor of oleamide hydrolaseas described in claim 1 represented by the following structure:##STR13##
 6. An inhibitor of oleamide hydrolase as described in claim 1represented by the following structure: ##STR14##
 7. An inhibitor ofoleamide hydrolase as described in claim 1 represented by the followingstructure: ##STR15##
 8. An inhibitor of oleamide hydrolase as describedin claim 1 represented by the following structure: ##STR16##
 9. Aninhibitor of oleamide hydrolase as described in claim 1 represented bythe following structure: ##STR17##
 10. An inhibitor of oleamidehydrolase as described in claim 1 represented by the followingstructure: ##STR18##
 11. An inhibitor of oleamide hydrolase as describedin claim 1 represented by the following structure: ##STR19##
 12. Aninhibitor of oleamide hydrolase as described in claim 1 represented bythe following structure: ##STR20##
 13. An inhibitor of oleamidehydrolase as described in claim 1 represented by the followingstructure: ##STR21##
 14. An inhibitor of oleamide hydrolase as describedin claim 1 wherein said head group is a fatty acid ester and includestwo or more electrophilic carbonyls and is selected from a groupconsisting of radicals represented by the following structures:##STR22## and wherein said hydrocarbon tail includes at least oneunsaturation.